Lightning Talk & Poster Australasian Extracellular Vesicles Conference 2018

Surfaceome of exosomes secreted from the colorectal cancer cell line SW480 (#54)

Rong Xu 1 , David Greening 1 , Maoshan Chen 1 , Alin Rau 1 , Adnad Shafiq 1 , Nobuhiro Takahashi 2 , Richard Simpson 1 2
  1. Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia
  2. Global Innovation Research Organization, Tokyo University of Agriculture and Technology, Tokyo, Japan

Exosomes are bidirectional cell-cell communicators that play a crucial role in both normal and pathological physiology. Although exosomal surface membrane proteins (surfaceome) enable target cell recognition and are an attractive source of disease markers they are still poorly understood. Here, we describe a comprehensive surfaceome analysis of exosomes secreted by the colorectal cancer (CRC) cell line SW480 using a combination of sodium carbonate extraction / Triton X 114 phase separation approach as well as mild proteolysis of intact exosomes using proteinase K (PK). Label-free quantitative mass spectrometry revealed  889 proteins of which 181 were predicted to be integral membrane proteins (IMPs) according to transmembrane hidden Markov model (TMHMM) analysis and hydrophobicity distribution using Grand Average of hydropathy (GRAVY) scores. Interrogation of the UniProt database predicted 104 peripherally-associated membrane proteins (PMPs). Surprisingly, 106 RNA-binding proteins (RBPs)/ RNA nucleoproteins (RNPs) were found in the carbonate/Triton X-114 insoluble fraction. Mild PK treatment of SW480-GFP labelled-exosomes revealed 31 proteolytically cleaved IMPs and 14 exoplasmic PMPs (e.g., CLU/ GANAB/LGALS3BP). A striking finding was 16 RBPs/RNPs (e.g., EIF3L/ RPLP0/SF3B3) appear bound to the outer exosome surface since they were sensitive to PK proteolysis. Our finding that outer surface-localised miRNA Let-7a-5p is RNase A resistant, but degraded by a combination of RNase A/PK treatment suggests exosomal miRNA species also reside on the outer surface of exosomes bound to RBPs/RNPs.